Biology subjects

Cote, C.

Publications and source records attributed to Cote, C..

6 recordsLinked to original sources

bioRxiv2026

Multiplexed enrichment and tracking of lineages with CloneSweeper

A fundamental challenge in studying therapy resistance is understanding whether it results from pre-existing cellular states ("priming") or drug-induced changes ("adaptation"). While lineage barcoding enables retrospective analysis of cells before and after treatment, current methods struggle to efficiently capture rare lineages in single-cell RNA sequencing (scRNA-seq) or isolate multiple specific lineages simultaneously for functional study. To overcome these limitations, we developed CloneSweeper, a multiplexed lineage tracking platform that pools enrichment libraries to isolate or enrich multiple rare lineages. CloneSweeper utilizes a dual-function barcode expressed as both a Cas9 gRNA for live-cell sorting and a 3 UTR transcript for high-recovery detection in 10x Genomics scRNA-seq. We applied CloneSweeper to a model of BRAF V600E melanoma, where we identified that resistance to targeted therapy emerges from a polyclonal population of rare, pre-existing lineages. By simultaneously targeting and enriching 21 distinct rare lineages prior to treatment, we defined a heritable, primed state characterized by de-differentiation and elevated mesenchymal markers. We demonstrate that these primed cells are not quiescent but instead exhibit upregulated inflammatory and stress response signaling, specifically via the AP-1 and NF-{kappa}B1 pathways. CloneSweeper thus provides a robust framework for dissecting the molecular mechanisms of rare biological phenomena through simultaneous, multiplexed lineage isolation.

bioengineering↗

bioRxiv2025

Transgenic Inducible MHC I Overexpression in Mouse Alveolar Type 2 Cells

The major histocompatibility complex class I (MHC I) is crucial in adaptive immunity, enabling CD8+ T cells to detect and eliminate infected and cancerous cells. Recent studies have uncovered significant variability in MHC I expression across tissues, challenging the traditional belief of uniform expression. Lung epithelial cells (LECs) express meager amounts of MHC I, which preserves the lung epithelium from excessive inflammation but renders it more susceptible to cancer and infection. Despite MHC I overexpression in various immunopathologies, its precise role in disease initiation or progression remains unclear due to the absence of suitable in vivo models for studying MHC I overexpression. This study introduces a novel mouse model with targeted surface MHC I upregulation. Leveraging a conditional Cre-lox system, we augmented Nlrc5 expression to specifically upregulate MHC I in alveolar type 2 (AT2) LECs, known for their low basal expression of MHC I and significant overexpression in disease. Our model demonstrated a rapid and sustained tenfold increase in MHC I surface expression persisting for up to a year without triggering pathology or inflammation. Comprehensive characterization and validation of this model indicated that MHC I overexpression does not serve as a primary initiator of respiratory diseases under steady-state conditions and shows a therapeutic window for increasing MHC I without significant damage to the lung epithelium. This adaptable model offers insights into the effects of tissue-specific MHC I regulation and presents new avenues for therapeutic development.

molecular biology↗

bioRxiv2023

Transposable elements regulate thymus development and function

Transposable elements (TE) are repetitive sequences representing [~]45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTEC). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multi-omic analyses of TEs in human and mouse thymic cells to elucidate their role in T cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDC). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN LJ/{beta}. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.

immunology↗

bioRxiv2021

Systematically quantifying morphological features reveals constraints on organoid phenotypes

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoids morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoids morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

systems biology↗

bioRxiv2020

Retrospective identification of rare cell populations underlying drug resistance connects molecular variability with cell fate

Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA FISH to directly capture rare cells that give rise to cellular behaviors of interest. Applied to BRAFV600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of [~]1:1000-1:10,000 cells) and relative persistence of MAP-kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences between several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones following drug treatment. Similarly, we show that treatments that modify the frequency of resistance can allow otherwise non-resistant cells in the drug-naive population to become resistant, and that these new populations are marked by the variable expression of distinct genes. Together, our results reveal the presence of hidden, rare-cell variability that can underlie a range of latent phenotypic outcomes upon drug exposure.

systems biology↗

bioRxiv2018

CRISPR/Cas9 Targeted Capture Of Mammalian Genomic Regions For Characterization By NGS

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines, and compare it to popular target-enrichment methods and to whole genome sequencing (WGS). The CRISPR/Cas9-based techniques allow for amplification-free capture of genomic regions, which reduces the possibility of sequencing artifacts. Other advantages of these methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of longer sequencing templates for real-time sequencing. The utility of these protocols has been proven by identification of transgene integration sites and flanking sequences in a number of CHO cell lines. However, data produced by these and other targeted capture methods are not always sufficient to analyze complex genomic rearrangements (CGRs) or unexpected sequences introduced into genome by vector integration events. In contrast, WGS provides complete information about vector integration sites, vector copy number, CGRs, and foreign DNA-but despite these benefits, WGS is not easily implemented due to the cost and complexity of the analysis.

genomics↗